Cytochrome P450s are the main enzymes responsible for the oxidative and reductive metabolism of numerous endogenous and xenobiotic substrates. The ability of the P450s to catalyze multiple types of reactions and their broad substrate specificity make these enzymes an excellent choice for engineering efforts to create new substrate specificities. We hope to engineer CYP119, the first thermophilic P450 to be identified thus far, into a self-sufficient P450 capable of metabolizing non-natural substrates. CYP119 was originally cloned from Sulfolobus solfataricus, an aerobic, acidothermophilic archaeon, and identified as a potential P450 based on its sequence homology to other members of the P450 superfamily. Because of its acidophilic and thermophilic properties, CYP119 is a more versatile model system than those currently available. A homology model of CYP119 has been constructed based on alignments with the four crystallized P450s. Using MidasPlus, important active site residues have been identified and mutated. Initial studies with T213 mutants indicate a prominent role for this residue in the specificity and the activity of CYP119. In addition, we are preparing to run MD simulations on wild-type and mutant CYP119 to investigate the impact of temperature on active site topology. DOCK is also an important tool that is helping to refine our understanding of P450 substrate specificity. DOCK has been tested with wild-type P450cam and two of its mutants. Further studies with a wider range of P450s and potential substrates are still required to make this approach of practical utility. We hope that the combined results from studies with P450cam, P450terp, P450eryf, P450BM-3 and CYP119 will provide the necessary information for the correct prediction of mutations that will effect desired changes in the catalytic specificity of P450s.